1. Field of the Invention
This invention relates to a .beta.-amylase with an improved thermostability as well as an improved enzyme stability in the alkaline pH region, a gene coding the enzyme and an expression vector containing the gene.
2. Description of the Related Art
Barley .beta.-Amylase
Barley .beta.-amylase is a .beta.-amylase (1,4-.alpha.-D-glucan maltohydrolase EC 3.2.1.2!) obtained from barley seeds and is well known along with soybean .beta.-amylase, as a useful enzyme for the industrial maltose production used for transfusional solutions and foodstuffs.
However, since barley is one of the principal agricultural products for the production of livestock feeds and beverages (such as beer and whisky), from the viewpoint of the global food situation in the future it is not advisable to consume the harvested barley as a source of .beta.-amylase.
Therefore, the method for producing .beta.-amylase in microorganisms using genetic engineering techniques has been given attention as an other source of this enzyme than the barley. If the efficient expression of the barley .beta.-amylase gene in a microorganism is accomplished, the steady supply of inexpensive .beta.-amylase will become possible, obviously contributing a great deal to the maltose production.
Gene of Barley .beta.-Amylase
As to the barley .beta.-amylase gene, the cDNA consisted of 1754 base pairs of cultivar Hiproly has been reported, and also the amino acid sequence consisted of 535 residues has been deduced (Eur. J. Biochem., 169, 517 (1987)). In addition, the cDNA consisted of 1775 base pairs of cultivar. Haruna Nijo has been reported, and also the amino acid sequence consisted of 535 residues has been established (J. Biochem., 115, 47 (1994)).
In studies on .beta.-amylase of cultivar Haruna Nijo, the expression vector (pBETA92) was already constructed by inserting a DNA fragment, which was prepared by deleting 55 base pairs of a full-length cDNA from its 5'-terminus and linking a SmaI linker, into the SmaI site of plasmid pKK223-3 (Pharmacia Biotech). Also the production of recombinant .beta.-amylase has been accomplished by transforming Escherichia coli JM109 (Toyobo) with said expression vector and expressing the recombinant .beta.-amylase gene therein. Furthermore, it was reported that the recombinant .beta.-amylase comprising 531 amino acids showed almost the same properties as barley .beta.-amylase (JP Hei6-58119; JP Hei6-303988).
However, a production of recombinant .beta.-amylase in microorganisms which shows almost the same properties to those of .beta.-amylase from barley seeds is not sufficient for the purpose. It is because of the fact that, since soybean .beta.-amylase is superior a little more to barley .beta.-amylase in the thermostability, soybean .beta.-amylase is more widely used in practice. Therefore, in order to improve the utility value of the barley .beta.-amylase, it is necessary to provide it at least with the similar function (thermostability) to that of soybean .beta.-amylase.
As to the barley recombinant .beta.-amylases with improved thermostability by protein engineering, it has been proved that double-mutant .beta.-amylase wherein Ser.sub.291 of the enzyme is replaced with Ala and Ser.sub.346le with Pro by site-directed mutagenesis is superior to the original recombinant .beta.-amylase (JP Hei6-126151).
To further improve the utility value of recombinant .beta.-amylase, it is necessary to construct .beta.-amylase with a further improved thermostability by protein engineering.